Efficacious vaccines against bordetella pertussis comprising a combination of individually purified pertussis antigens

ABSTRACT

This invention is directed to a vaccine for the prevention of disease caused by Bordetella pertussis which comprises the pertussis antigens filamentous hemagglutinin, detoxified lymphocytosis promoting factor and a 69 kilodalton outer membrane protein, where said antigens are individually purified prior to being combined to form the vaccine. The invention is further directed to pertussis vaccines where the antigens are combined in any ratio, including ratios not possible in whole cell or co-purified acellular pertussis vaccines. The pertussis antigens may be further combined with other individually purified pertussis antigens, pertussis structural components, adjuvants, stabilizers and non-pertussis vaccine components.

This is a divisional of application Ser. No. 07/549,236, filed on Jul.11, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to vaccines efficacious against Bordetellapertussis which are prepared by individually purifying specificpertussis antigens which are then combined to form the vaccine. Inaddition, the vaccine may contain pertussis structural components andnon-pertussis vaccine components.

BACKGROUND OF THE INVENTION

The bacterium Bordetella pertussis is the causative agent of pertussisor whooping cough, a serious and potentially fatal infectious disease ofthe upper respiratory tract. Pertussis vaccines currently used containchemically inactivated whole cells of B. pertussis. More recently,acellular pertussis vaccines were developed which are based on materialobtained by chemical and physical fractionation of B. pertussiscultures.

Whole cell vaccines contain the antigenic components necessary toprovide protection from pertussis disease and their efficacy in humansis generally well accepted. However, whole cell vaccines also containcomponents which are not required for protection. Some of thesecomponents, such as endotoxin, have been implicated in undesired effectswhich may occur coincident with pertussis immunization (Bibliography 1).

Acellular vaccines are less complex than whole cell vaccines, becausethey lack endotoxin, DNA, and cellular components not associated withprotection, such as extraneous enzymes and other proteins. However,acellular vaccines, such as that developed by Takeda ChemicalIndustries, Ltd., Osaka, Japan (2) and the Kanonji Institute, TheResearch Foundation for Microbial Diseases (Biken) of Osaka University,Japan (3), may also contain more components than necessary to conferprotection. In addition, some acellular vaccines are tedious to produce,because of their requisite co-purification procedure (1).

Based on animal protection studies, several B. pertussis antigens havebeen proposed as protective antigens, namely, lymphocytosis promotingfactor (LPF, also known as pertussis toxin, which is detoxified beforeuse and is thereafter referred to as pertussis toxoid) (1), histaminesensitizing factor, or islet activating factor (4), filamentoushemagglutinin (FHA) (1), agglutinogens such as fimbriae (5), and outermembrane proteins (1), such as the 69 kilodalton (69K) outer membraneprotein (6).

Each of these antigens is able to individually protect animals in one ormore animal models. However, animal models are of limited use inpredicting efficacy of a pertussis vaccine in humans, because the B.pertussis organism is a natural pathogen only in humans (7).

Based on animal data, two acellular vaccines developed by Biken, onecontaining LPF and FHA, and one containing LPF alone were evaluated inhumans (8,9). Overall, the efficacies of these two vaccines were lowerthan that of whole cell vaccines. The acellular Biken vaccines were only58-69% (LPF and FHA) or 41-55% (LPF alone) efficacious in conferringprotection as compared to the 85-95% efficacy of the whole cell vaccine(8,9). This indicates that other antigens may need to be included toobtain efficacious pertussis vaccines.

SUMMARY OF THE INVENTION

The vaccines of this invention do not contain undesired or extraneouscomponents. In contrast, existing whole cell pertussis vaccines,although generally efficacious, contain undesired components. Currentacellular pertussis vaccines, which are also generally efficacious, maycontain more components than are necessary to confer protection.

Accordingly, it is an object of this invention to describe efficaciouspertussis vaccines which are prepared by individually purifying specificpertussis antigens and then combining the purified antigens to form thevaccine. At a minimum, the vaccine comprises FHA, LPF and the 69Kprotein.

It is a further object of this invention to describe pertussis vaccineswherein the antigens are combined in any ratio to optimize protection.Thus, the vaccines can have ratios not possible in whole cell orco-purified acellular pertussis vaccines.

It is yet another object of this invention to include additionalindividually purified pertussis components in the vaccine, such as the30 kilodalton outer membrane protein and/or one or more agglutinogenssuch as fimbriae.

It is an additional object of this invention to further improve theefficacy of the above vaccine by including other pertussis structuralcomponents. The pertussis antigens may be conjugated to each other or topertussis structural components.

It is a still further object of this invention to combine the abovevaccine with non-pertussis vaccine components to create multivalentvaccines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pertussis vaccines efficacious inhumans which are well suited to commercial scale production, and whichrepresent a significant improvement over the currently available wholecell and acellular vaccines. Purification of specific antigensindividually with combination afterwards has several advantages.

Antigens can be combined in any desired ratio, including ratios notpossible in whole cell or co-purified acellular vaccines. Thus, anantigen ratio can be chosen which gives the optimal response in humans.This is in contrast to the acellular vaccines which, as fractionatedantigen mixtures, have basically a fixed antigen ratio. The Biken twocomponent vaccine has a FHA:LPF ratio of 50:50 (total 100 parts). TheTakeda vaccine has a FHA:LPF:69K:agglutinogen ratio of 85:7:7:1.Throughout this application, ratios are based on the proportions ofmicrograms of antigens contained in a given dose of vaccine. Whole cellvaccines are also limited in the ratios of antigens to the rangespresent in the organisms used.

Chemical detoxification procedures which are needed to remove undesiredbiological activities of LPF can be performed selectively on the LPFantigen alone prior to combining the antigens into a vaccine. Theremaining antigens, which do not require a detoxification procedure, arethus not unnecessarily exposed to the chemicals used for detoxificationwhich could reduce their effectiveness as antigens by destroying theirstructure or conformation which is responsible for their ability toconfer protection. In currently available co-purified acellularvaccines, on the other hand, all antigens are subjected to a chemicaldetoxification procedure. The resulting vaccine of this invention doesnot contain undesired components such as endotoxin.

When considering vaccine production on a practical commercial scale, thenumber of antigens in a pertussis vaccine has to be limited. Dependingon the effort required for the purification of the individual antigens,three to four antigens may be a limit. It is, however, feasible thatinclusion of further antigens might increase vaccine efficacy to somedegree.

The antigens included in the vaccines of this invention are identifiedby analyzing the composition of the Takeda acellular pertussis vaccinedescribed above, which has been found to have good efficacy (81-95%) inhumans (10).

In one embodiment of this invention, the vaccine includes LPF, in itsnon-toxic form (pertussis toxoid), FHA and the 69K protein. In anotherembodiment of this invention, the vaccine includes LPF, FHA, the 69Kprotein and a 30 kilodalton (30K) outer membrane protein (11). Inaddition, agglutinogens such as fimbriae can be included in either ofthese vaccines.

The individual pertussis components are either purified from native B.pertussis using purification procedures which are known in theliterature and exemplified below, or through other methods such as theuse of recombinant DNA techniques to express the individual components.The antigens are expressed in a suitable host organism such as membersof the genus Bordetella, E. coli, Haemophilus, Streptomyces spec.,Bacillus subtilis, yeast, insect (such as baculovirus) or mammalian celllines, or any other suitable host expression system.

The LPF component is detoxified using a chemical detoxificationtechnique as described in the literature (12,13,14). Alternatively, LPFmay be detoxified by genetic means through the introduction ofsite-directed mutations which abolish the undesired biologicalactivities of the toxin (15,16).

The individually purified antigens are then blended in the desired ratioto produce the vaccine. The vaccines of this invention may also includephysiologically acceptable pertussis structural components. Thesecomponents may be included for the purpose of avoiding side effects orto enhance the presentation of the antigenic components and therebyimprove the efficacy of the vaccine. Examples of such structuralcomponents include polysaccharides, lipopolysaccharides, lipids,proteins, glycoproteins and lipoproteins.

The pertussis antigens may be conjugated to each other or to pertussisstructural components using conventional techniques. The components maybe conjugated directly by reductive amination as described by Anderson(17). Alternatively, the components may be linked through a spacerelement such as adipic acid dihydrazide as described by Gordon (18) or6-aminocaproic acid as described by Hilleman et al. (19).

Other non-pertussis vaccine components can be added by conventionaltechniques to prepare multivalent vaccines. Multivalent vaccines aredesirable, particularly for infants and small children, in that theyreduce the number of dosage administrations needed to confer protectionagainst a series of disease organisms. Examples of such other vaccinecomponents include diphtheria toxoid, tetanus toxoid, inactivated polioviruses, Haemophilus influenzae, Haemophiluspolyribosylphosphate-protein conjugates, Neisseria meningococcus,Pneumococcus and hepatitis B.

If desired, the vaccine may also include a pharmaceutically acceptableadjuvant such as aluminum gels, calcium gels, modified muramyldipeptides, monophosphoryl lipid A, liposomes, time release capsules,polyglycolic acids and polyamino acids. Polyglycolic acids and polyaminoacids are also useful for oral delivery of the vaccine. Examples ofaluminum gels useful as adjuvants include precipitated aluminum saltssuch as aluminum phosphate and aluminum hydroxide. Suitablepreservatives such as thimerosal, dextran and glycerine can be added tostabilize the final vaccine. If it is desired that the vaccine be ininjectable form, immunologically acceptable diluents or carriers may beincluded in a conventional manner to prepare liquid solutions orsuspensions.

The vaccines of this invention are administered by conventional meanssuch as parenteral injection (subcutaneous or intramuscular), as well asby oral or intradermal administration into human beings to elicit anactive immune response for protection against infection caused by B.pertussis. The dosage to be administered is determined by means known tothose skilled in the art.

In order that this invention may be better understood, the followingexamples are set forth. The examples are for the purpose of illustrationonly and are not to be construed as limiting the scope of the invention.

EXAMPLE 1 Purification of LPF

LPF is isolated from the culture supernatant of a B. Pertussis cultureusing conventional separation methods. For example, using the methoddescribed by Sekura et al. (20), LPF is isolated by first adsorbingculture supernatant onto a column containing the dye-ligand gel matrix,Affi-Gel Blue (Bio-Rad Laboratories, Richmond, Calif.). LPF is elutedfrom this column by high salt, such as 0.75M magnesium chloride and,after removing the salt, is passed through a column of fetuin-Sepharoseaffinity matrix composed of fetuin (Gibco Laboratories, Grand Island,N.Y.) linked to cyanogen bromide (CNBr)-activated Sepharose 4B(Pharmacia, Piscataway, N.J.). LPF is eluted from the fetuin columnusing 4M magnesium salt either stepwise or with a gradient. Theresulting preparation is essentially free from endotoxin and yields abiologically active protein which can be assayed by hemagglutinationassay (HA), ADP-ribosylation assay, CHO cell toxicity assay or otherconventional methods suitable for determining the biological activity ofLPF.

Alternatively, using the method of Irons et al. (21), culturesupernatant is adsorbed onto a CNBr-activated Sepharose 4B column towhich haptoglobin is first covalently bound. The LPF binds to theadsorbent at pH 6.5 and is eluted from the column using 0.1M Tris/0.5MNaCl buffer by a stepwise change to pH 10.

EXAMPLE 2 Detoxification of LPF

LPF purified by either method described in example 1, is then detoxifiedto remove undesired activities which could cause side reactions of thefinal vaccine. Any of a variety of conventional chemical detoxificationmethods can be used such as treatment with formaldehyde, hydrogenperoxide, tetranitro-methane, or glutaraldehyde.

For example, LPF can be detoxified with glutaraldehyde using amodification of the procedure described in Munoz et al. (12), such thatthe residual biological activities do not pose any safety concern, whilethe desired immunogenic properties of LPF are still retained. PurifiedLPF is placed in a solution containing 0.01M phosphate buffered saline(PBS) (instead of the solution of 20 mM sodium phosphate with 0.5M NaCl(pH 7.6) used in Munoz). The solution is treated with enough 0.2%glutaraldehyde solution made in the same buffer to bring theconcentration of glutaraldehyde to 0.05%. The mixture is incubated atroom temperature for two hours, and then enough 0.2M L-lysine solution(made in the same buffer) is added to bring the concentration ofL-lysine to 0.02M. The mixture is further incubated for two hours atroom temperature and then dialyzed for two days against 0.01M PBS(instead of the 20 mM sodium phosphate buffer containing 0.5M NaCl and0.02M L-lysine (pH 7.6) used in Munoz) to complete the detoxification.

If recombinant techniques (15,16) are used to prepare a LPF mutantmolecule which shows no or little biological activity, a milder form ofthese chemical procedures can be used. For example, a shorter exposureto the chemical, reduced concentration of the chemical, ordetoxification at lower temperature may be possible. In some instances,the detoxification step can be omitted altogether.

EXAMPLE 3 Purification of FHA

FHA is purified from the culture supernatant using a modification of thepublished method of Cowell et al. (22). Growth promoters such asmethylated beta-cyclodextrins may be used to increase the yield of FHAin culture supernatants. The culture supernatant is applied to ahydroxylapatite column. FHA is adsorbed onto the column, but LPF is not.The column is extensively washed with Triton X-100 to remove endotoxin.FHA is then eluted using 0.5M NaCl in 0.1M sodium phosphate and, ifneeded, passed through a fetuin-sepharose column to remove residual LPF.Additional purification can involve passage through a Sepharose Cl-6Bcolumn (Pharmacia, Piscataway, N.J.). FHA is sterilized by passing itthrough a suitable filter, such as Millex (Millipore Corporation,Bedford, Mass.).

Alternatively, FHA may be purified using monoclonal antibodies to theantigen, where said antibodies are affixed to a CNBr-activated affinitycolumn (23).

EXAMPLE 4 Purification of 69K Outer Membrane Protein

The 69K outer membrane protein is recovered from the bacterial cells,using the procedure described in co-pending, commonly-assigned U.S. Ser.No. 448,777, filed Dec. 11, 1989, which is hereby incorporated byreference.

In summary, the 69K protein is recovered from the bacterial cells byfirst inactivating the cells with a bacteriostatic agent such asthimerosal. The inactivated cells are suspended in an aqueous mediumsuch as PBS (pH 7-8) and subjected to repeated extraction at elevatedtemperature (45°-60° C.) with subsequent cooling to room temperature or4° C. The extractions release the 69K protein from the cells. Thematerial containing the 69K protein is collected by precipitation andpassed through an Affi-Gel Blue column. The 69K protein is eluted with ahigh concentration of salt, such as 0.5M magnesium chloride. Afterdialysis, it is passed through a chromatofocusing support such as aPolybuffer Exchanger Gel PBE 94 column (Pharmacia). The recovered 69Kfractions are sterilized by filtration through a suitable filter such asMillex.

EXAMPLE 5 Preparation of Vaccine

To prepare a final vaccine product of this invention, the purified andsterilized pertussis components prepared according to Examples 1-4 arefirst combined in aqueous medium containing sodium chloride and sodiumphosphate. Then, if desired, aluminum chloride and sodium hydroxide areadded in presence of thimerosal preservative, resulting in an adsorbedvaccine which is formed in situ. Alternatively, a preformed adjuvant maybe added to the active components, either separately or after thecomponents are combined. In either procedure, if necessary the pH isadjusted with sodium hydroxide to a desired value in the range pH6.3-7.5. In a preferred embodiment of this invention, the final vaccinehas an FHA:LPF:69K ratio of 57:29:14 (or 4:2:1 to be precise).

EXAMPLE 6 Purification of 30K Outer Membrane Protein

The 30K outer membrane protein is recovered from B. pertussis cells byprocedures described in the literature (11). In summary, B. pertussiscells are inactivated by thimerosal, suspended in an aqueous solutioncontaining 2M sucrose, 0.1M Tris hydrochloride (pH 7.8), 1% sodium EDTA(pH 7.0) and 1.0% lysozyme. The suspension is incubated at 30 ° C. forone hour. DNase is added to reduce the viscosity which increases due tocell lysis. Centrifugation of the suspension at 20,000×g for one hour at30° C. gives a supernatant which is adjusted to pH 5.0 with 0.2M HCl andstored overnight at 4° C. to precipitate out a mixture of proteins. Theprecipitate is collected by centrifugation at 30,000×g for 15 minutes.

Extraneous materials are removed by adding 2% Triton X-100 in water andmixing, followed by the addition of ethanol. The mixture is incubatedovernight at 4° C. and then centrifuged at 9,000×g for 20 minutes. Theprecipitate is washed with distilled water and the solution is dialyzedfor three days against PBS. The 30K protein is then obtained bysuccessive DEAE-Sepharose Cl-6B ion exchange chromatography (in a TrisHC1, EDTA, Zwittergent (TEZ) buffer using a 0 to 1M NaCl gradient forelution) and Sephacryl S-300 gel filtration chromatography (eluting witha TEZ buffer). The 30K protein is sterilized by passage through asuitable filter. The purified 30K protein may then be combined with thecomponents described in Examples 1-4 to form a final vaccine product ofthis invention using the procedure set forth in Example 5.

BIBLIOGRAPHY

1. Manclark, C. R., et al., pages 69-106, in Bacterial Vaccines,Germanier, R., ed., Academic Press (1984).

2. U.S. Pat. No. 4,455,297.

3. Sato, Y., et al., The Lancet, 122-126 (Jan. 21, 1984).

4. Yajima, M., et al., J. Biochem., 83, 295-303 (1978).

5. Cowell, J. L., et al., Infection and Immunity, 55, 916-922 (1987).

6. Shahin, R. D., et al., "Immune Protection Mediated by the 69K OuterMembrane Protein of Bordetella pertussis", page 51, Abstracts of the89th Annual Meeting of the American Society for Microbiology (1989).

7. Kimura, A., et al., Infection and Immunity, 55, 7-16 (1990).

8. Marwick, C., JAMA, 259, 2057-2059 (1988).

9. Kallings, L. O., The Lancet, 955-960 (Apr. 30, 1988).

10. Mortimer, E. A., Jr., The Tokai Journal of Expt'l and ClinicalMedicine, 13 Supp., 29-34 (1988).

11. Monji, N., et al., Infection and Immunity, 51, 865-871 (1986).

12. Munoz, J. J., et al., Infection and Immunity, 33, 820-826 (1981).

13. Relyveld, E. H., et al., Methods in Enzymology, 93, 24-60 (1983).

14. Kaslow, H. R., et al., Biochemistry, 26, 4397-4402 (1987).

15. Zealey, G., et al., Vaccines '89, 259-263 (1989).

16. Pizza, M., et al., Science, 246, 497-500 (1989).

17. U.S. Pat. No. 4,673,574.

18. U.S. Pat. No. 4,496,538.

19. U.S. Pat. No. 4,459,286.

20. Sekura, R. D., et al., J. Biol. Chem., 258, 14647-14651 (1983).

21. Irons, L. I., et al., Biochimica et Biophysica Acta, 580, 175-185(1979).

22. Cowell, J. L., et al., Seminar in Infectious Diseases, 4, 371-379(1982).

23. Selmer, J. C., Acta Path. Microbiol. Immunol. Scand. Sect. C, 92,279-284 (1984).

We claim:
 1. A multivalent vaccine which comprises:(a) a vaccineefficacious in preventing disease caused by Bordetella pertussis,wherein antigens of said vaccine comprise Bordetella antigens consistingof: Bordetella pertussis antigens filamentous hemagglutinin (FHA),detoxified lymphocytosis promoting factor (LPF) and 69 kilodalton outermembrane protein, and wherein said pertussis antigens are individuallypurified prior to being combined to form said vaccine, and (b)diphtheria toxoid and tetanus toxoid vaccines.
 2. The multivalentvaccine of claim 1 which further comprises at least one of an adjuvant,diluent or carrier.
 3. The vaccine of claim 2 wherein the adjuvant is aprecipitated aluminum salt such as aluminum phosphate or aluminumhydroxide.
 4. The multivalent vaccine of claim 1 which further comprisesat least one of a stabilizer or preservative.
 5. The vaccine of claim 4wherein the stabilizer is thimerosal.